BIOCHEMICAL

Vol. 75, No. 3, 1977

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

PREFERENTIAL DEGRADATION OF NEWLY SYNTHESIZED RIBOSOMAL PROTEINS IN RAT LIVER TREATED WITH A LOW DOSE OF ACTINOMYCIN D Kunio Tsurugi Department

and Kikuo

Ogata

of Biochemistry,

Niigata,

Japan

Received

January

26,

Niigata

University

School

of Medicine,

1977

SUMMARY: The administration of a low dose of actinomycin D to partially hepatectomized rats, which selectively inhibited rRNA synthesis, caused the preferential degradation of newly synthesized ribosomal proteins in regenerating rat liver with an apparent half-life of about 20 to 40 min. Our previous was selectively stration mized

experiments inhibited

rats,

isotopic

amino acids

actinomycin the

liver

of newly

actinomycin

D-treated supported

treatment

resulted leucine

ribosomes ribosomal

regenerating extent

Copyright All rights

0

of

the

newly

(1) as well proteins

rat

liver

of the decrease

1977 by Academic reproducfion in my

Press,

form

1 h with

even at 5 min after possibilities of ribosomal

(B) the

proteins

rapid

in the

of our

livers

subseauent

since

of -in vivo

degraof ex-

actinomycin

incorporation

D of

ribosomal

proteins

as the decrease

in its

incorporation

postmitochondrial

However,

observed

Inc. reserved.

for

synthesized

by the (2).

extracted

following

possibility,

admini-

hepatecto-

synthesis

The results

former

the

the

and/or

ribosomal

in the decrease into

in vivo --

of

D treatment

rats.

by the

proteins

decreased

inhibition

synthesized

periments

labeled

We indicated

(A) the

liver

D to partially

was selectively

by actinomycin

dation

into

homogenate

when rRNA synthesis

rat

of ribosomal

D treatment.

decrease:

proteins

liver

in regenerating

the radioactivity

total

labeled

showed that

of a low dose of actinomycin

from the

for

(1)

the

in these

supernatant

finding experiments

that

on

from

the was rather

525 ISSN

0006-291X

BIOCHEMICAL

Vol. 75, No. 3, 1977

low,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

in the case of

especially

the

second possibility

ity

the

the cell-free

(B) described

experiments

reported

above.

system,

sugqested

To test

the possibil.-

here were carried

out.

MATERIALS AND METHODS: L-[3H]leucine (46.9 Ci/mmol) was obtained from the RadToaFCentre, Amersham, England. Partially hepatectomized rats 18 h after the operation (1) were injected intraperitoneally with 550 uq of actinomycin D per kg body weight (3) and 1 h later were used for the labeling experiments, As the control partially hepatectomized rats without actinomycin D treatment were used. Purificati-on procedures of ribosomal proteins from the homogenate of rat liver were as described previously (1, 4). Briefly, 10 volumes of acetone were added to the homogenate and the mixture stood at -20" overnight. From the precipitate after low speed centrifuqation ribosomal proteins were extracted with acetic acid and the acetic acid-soluble proteins were subjected to CM-cellulose column chromatography. Fraction I, eluted with a linear NaCl gradient from 0.25 to 0.35 M, contained the bulk of ribosomal proteins. Fraction P, eluted by lower concentrations of NaCl contain the bulk of cell sap proteins (4). In some experiments Fraction I was further purified by Sephadex G-200 column chromatography which yielded Component II, more than 85% of which was shown to consist of ribosomal proteins although it also contained newly synthesized histones and traces of cell sap proteins (4). The radioactivity of protein fractions was measured as described previously (1, 4). RESULTS AND DISCUSSION: proteins after

For measuring

we used cycloheximide pulse-labeling

liminary

of

experiment

to inhibit

rat

the

liver

rat

the total liver

protein

of

synthesis

complete

inhibition

what delayed.

on the

total

in the

The results

used for

the measurement

of the

proteins

in regenerating

rat

experiments After leucine

for

were carried actinomycin 15 min,

1, the

liver.

f3H]leucine

administration

and complete proteins,

that

rate

of

(5

of regenerating

of ribosomal

indicate

As a pre-

of cycloheximide

proteins

and ribosomal

of

synthesis

incorporation

rapid

of synthesis

rate

[3H]leucine.

As shown in Fig.

resulted

of the

protein

effects

and ribosomal

were examined.

of cycloheximide

with

inhibitory

mq per 100 g of body weight) into

the degradation

inhibition although

proteins

the

was some-

cycloheximide

may be

of degradation

of ribosomal

Therefore,

following

the

out. D-treated

one group

rats of rats

526

were labeled was sacrificed

with

L3Hl-

as 0 time

BIOCHEMICAL

Vol. 75, No. 3, 1977

time

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

after

c@oheximlde

treatment

(min.)

Fig. 1. Effects of cycloheximide treatment on the in vivo labeling of the total and ribosomal proteins of regeneratingrat liver. Rats at 18 h after partial hepatectomy received an intraperitoneal injection of 5 mg of cycloheximide per 100 g of body weight and after various periods 10 uCi of [3H]leucine per rat was administered. One hour later rats were sacrified and total ribosomal protein was prepared from EDTA-treated ribosomes and its specific activity was measured as described previously (1). The relative specific activity was expressed as % of the value of rats without cycloheximide treatment. e-r, total ribosomal protein; o------o, total liver protein.

control

and others

heximide-treated the

rats

administration

ribosomal

proteins

by Sephadex

at

activity

at least

for

decreased

half-life

cycloheximide.

were sacrificed ['Hlleucine.

activities point

as described

a half-life

which

are shown in Fig. protein

may be somewhat shorter,

527

homogenate

previously,

and the total 2.

While

since

40 min.

Component II

liver

the

almost

treatement, 20 to

finally

Component II.

remained

cycloheximide of about

liver

yielded

of Component II

of the total

Cyclo-

at 40 min and 60 min after From the

filtration

40 min after with

with

were purified

each time

specific

II

of

G-200 gel

The specific protein

were treated

constant

that

of Component

The actual is

contami-

Vol. 75, No. 3, 1977

BIOCHEMICAL

0

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

20

40

6o min.

Fig. 2. Time course of specific radioactivities of total and ribosomal proteins of actinomycin D-treated rat liver after administration of cycloheximide. Actinomycin D-pretreated rats received an intraperitoneal injection of 50 uCi of [3H]leucine per 100 g of body weight. After 15 min rats were injected with cycloheximide as described in Fig. 1. At the indicated times rats were sacrificed and Component II was prepared from the livers as described previously (4). The proteins of Component II were precipitated with trichloroacetic acid. The specific activities of the total liver protein and Component II were measured as described previously (4). *cycloheximide; l -, ribosomal proteins (component II); ototal liver protein.

nated tion

with

histones

of which

may not

be affected

To obtain

further

evidence

ribosomal

proteins

ing double the time rats

and traces

without

[14C] or other

for

of these

ratio

indicated.

3a.

rats with

rat

pretreated

cycloheximide

after

sacrificed

at

homogenate

at each time

liver

homogenate

from

Fraction

0 time

P and Fraction

528

the

followto

hepatectomized with

either

controls. with

point control

I which

The

actinomycin

labeling

specified

liver

of

according

were labeled as 0 time

and rats

liver,

out

Partially

D treatment

15 min and then

The l4 C-labeled with 3H-labeled

degradation

and sacrificed

D were administered leucine

preferential

for

were carried

actinomycin

degrada-

D treatment.

experiments shown in Fig.

the

by actinomycin

D-treated

[3H]leucine

group

sap proteins

in actinomycin

labeling schedule

of cell

with time

[ 14c1 points.

was mixed rats contained

at the the

Vol. 75, No. 3, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

3H-leucine

0)

llbmin. 0 14C - lyclne

1, Actinomycin 1

“C-leyoine.

D.

0.21 0

60min.

+ , 20

time control.

CpoP

4 0 d cl& y (3)

45 min.

40

t?Z) , 25min (4)

,

50 mln

radioactivities of ribosomal Fig. 3. Time courses of relative proteins of rat livers from actinomycin D-treated and control rats after cycloheximide treatment.Actinomycin D-treated (1 h) and control rats received an intraperitoneal injection of 25 uCi of [l4C]leucine per rat. After 15 min the rats were injected intraperitoneally with cycloheximide and sacrificed according to the time schedule shown in the upper panel (a). As the 0 time control, two control rats received an intraperitoneal injection of 200 uCi of f3H]leucine and were sacrificed after 15 min. r14c1 labeled liver homogenate at each time was combined with 0 time control liver homogenate in the ratio to make 3H to 14C ratio to be 3. Fractions P and I were prepared from the combined homogenate and subjected to one-dimensional acrylamide gel electrophoresis. The 14C to 3H ratios of the individual gel fractions from Fraction I were measured as described previously (1) and the relative Fraction I to P ratio at each time was expressed as described in the text. ok, actinomycin *cycloheximide; D-treated rat liver: o----o, control rat liver.

bulk

of cell

were prepared

sap proteins

and ribosomal

proteins,

from the combined homogenate.

then

subjected to one-dimensional acrylamide * 14 at pH 8.6. c to 3H-ratios of individual

respectively,

Both fractions

were

gel electrophoresis gel

fractions

were

corrected by multiplying by a suitable factor such that the * While the bulk of proteins of Fraction P runs to the anode, almost all ribosomal proteins run to the cathode at pH 8.6 (4).

529

Vol. 75, No. 3, 1977

average

BIOCHEMICAL

of the

ratios

of gel

and designated as corrected 14 c to 3H ratios corrected then

determined.

further

This

corrected

Since

sap protein (data

time

the

was less somal

degradation

rate

the

proteins

was inhibited

previously is

noticeable

Fraction

were not

earlier

into

proteins. In the

Fraction the

D-

could

of

by actinomycin

the show the

case of actino-

I to P ratio

rate

during

experiments,

of ribosomal

that

cell

D treatment

degraded

I to P ratio

3b.

to be I to P

amino acid

Fraction

that

almost

treatment

preferentially

the

linearly

at 45 min after

up to

control

at 0 time

synthesis

of ribo-

D treatment

Fraction

45 min after

rats

cycloheximide

inhibition

relative

in actinomycin

in the case of

delayed

control

was

as des-

(1).

decreased

while

time

by actinomycin

in the

relative

1, suggesting

It

labeled

are shown in Fig.

than

cribed

0 time

gel was

even in the case of actinomycin

relative

rats

on the

relative

sap proteins

of the

D-treated

of the

inhibited

as indicated

The results mycin

of

proteins

at each labeling

as the

slightly

period

liver

course

ratio

incorporation

time

rat

relative

average

shown) and cell

such a short treated

of ribosomal

was designated

was only

not

fractions from Fraction P became 1 3 14c to H ratio. The average of

so as to make that

1 and the ratio ratio.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

this

cycloheximide

D-treated ratio

treatment

of ribosomal

I to P ratio

protein

rat

liver,

increased

probably

slightly

due to the

synthesis

by cyclohexi-

mide. From these

results

ribosomal

proteins

unavailable

for

newly It

synthesized

it

may

be concluded

in actinomycin

the

assembly

D-pretreated

of

ribosomes

rRNA are rapidly

must be added that

after

that

degraded

the completion

530

newly rat

owing to

liver the

synthesized which lack

are

of

by unknown mechanisms. of this

work,

Phillips

Vol. 75, No. 3, 1977

and McConkey proteins

BIOCHEMICAL

suggested

in HeLa cells.

AND BIOPHYSICAL

such a degradation

RESEARCH COMMUNICATIONS

of unbound ribosomal

(5).

ACKNOWLEDGEMENT:This work was supported by a Grand-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan. REFERENCES (1) Tsurugi, K., Morita, T. and Ogata, K. (1972) Eur. J. Biochem. 2, 585-592 (2) Ogata, K., Tsurugi, K. and Nabeshima, Y. (1974) Acta biol. Med. germ. 33, 963-969 S.H. and Hoagland, M.B. (1967) Biochem. J. -'103 (3) Wilson, 556-566 K. and Ogata, K. (1976) J. Biochem. 79, 883-893 (4) Tsurugi, (5) Phillips, W.P. and McConkey, E.H. (1976) J. Bzl. Chem. 251, 2876-2881

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